1. Field of the Invention
The present invention is in the field of medicine, particularly in the field of monoclonal antibodies against human ghrelin useful for treatment of obesity and obesity-related disorders, including non-insulin dependent diabetes mellitus, Prader-Willi syndrome, hyperphagia, and impaired satiety. More specifically, the present invention relates to humanized, affinity matured monoclonal antibodies and antigen-binding portions thereof that preferentially bind acylated human ghrelin with respect to unacylated human ghrelin.
2. Description of Related Art
Obesity is a complex, chronic disease characterized by excessive accumulation of body fat, and has a strong familial component. Obesity increases the risk of illness from about 30 serious medical conditions, including osteoarthritis, Type II diabetes, hypertension, cancer, and cardiovascular disease, and is associated with increases in deaths from all causes. Additionally, obesity is associated with depression, and can further affect the quality of life through limited mobility and decreased physical endurance.
Human ghrelin is a recently identified peptide hormone having the amino acid sequence GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID NO:1) that, when acylated at the serine at amino acid position three (Ser3) with an n-octanoyl group (“C8” or “C8 acylated ghrelin”), binds the growth hormone secretagogue receptor (GHS-R1a), resulting in release of growth hormone (Kojima et al., Nature 402:656-660, 1999).
Ghrelin has been demonstrated to lead to fat deposition when administered to mice (Tschop et al., Nature 407:908-913, 2000). Ghrelin is synthesized primarily in the stomach, and its levels increase during food deprivation in animals (Kojima et al., Nature 402:656-660, 1999) and peak prior to eating in humans (Cummings et al., NEJM, 346:1623-1630, 2002). Recently, it has been shown that persons who underwent gastric bypass surgery and lost up to 36% of their body weight had greatly reduced ghrelin levels, with loss of pre-meal peaks in ghrelin secretion. Persons with Prader-Willi syndrome, a genetic disorder that causes severe obesity with uncontrollable appetite, have extremely high levels of ghrelin (Cummings et al., NEJM, 346:1623-1630, 2002). These observations indicate that ghrelin plays a key role in motivating feeding.
In addition to its role in eating disorders, ghrelin has also been shown to have a proliferative effect in the HepG2 hepatoma cell line (Murata et al., J. Biol. Chem., 277:5667-5674, 2002) and in prostate cancer cell lines (Jeffrey et al., J. Endocrinol., 172:7-11, 2002; Yeh et al. (Clin. Cancer Res., 11(23):8295-8303, 2005). The growth of other cell types including, for example, H9c2 cardiomyocytes, pancreatic adenocarcinoma, adrenal cells, pituitary somatotroph cells, adipocytes, osteoblastic cells, breast cancer cell lines, etc., is also enhanced by ghrelin (discussed in Yeh et al., supra).
International application PCT/US2004/014537 (WO 2005/016951) discloses the Fab portion of a murine monoclonal antibody to the N-terminal end of acylated ghrelin, designated therein as Fab 1111. This Fab, which binds between amino acids 1 and 8 of C8 acylated human ghrelin with a Kd of 1.04 nM, is a murine sequence referred to herein as a C2 Fab. The amino acid sequences of the heavy chain and light chain of C2 murine Fab 1111 are shown below, with the CDR regions underlined:
C2 Ghrelin Murine Fab 1111Heavy Chain(SEQ ID NO:2)EIQLQQSGAELMKPGASVKLSCKATGYIFTGYWIEWVKQRPGHGLEWIGE ILPGSGSTNYNEKFKGKATFTADTSSNTANMQLSSLTTEDSAIYYCARYP QFRLRRERIAYWGQGTLVTVSVAKTTPPSVYPLA Light Chain(SEQ ID NO:3)DLVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKL LIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPY TFGAGTKLELKR.
There is a pressing therapeutic need for a means to treat obesity and obesity-related disorders. Due to its potential role in inducing feeding, ghrelin is a desirable target for therapeutic intervention. In particular, a monoclonal antibody against ghrelin that preferentially binds the acylated form of ghrelin with respect to the unacylated form of ghrelin may provide such a therapy. Of particular importance therapeutically is a humanized, affinity-optimized form of such a monoclonal antibody. Such an anti-ghrelin antibody may be useful for the treatment of obesity, including morbid obesity, and related disorders including, for example, Type II non-insulin dependent diabetes mellitus (NIDDM), Prader-Willi syndrome, hyperphagia, and impaired satiety.
Therapeutically beneficial antibodies that bind to the epitope recognized by the C2 antibody will desirably be stable in solution, display favorable pharmacokinetics, and possess improved affinity toward an epitope formed by amino acids 1 to 8 of acylated ghrelin. Therefore, there is a need in the art for humanized antibodies having characteristics similar to or better than C2 murine Fab 1111, which will be therapeutically efficacious in humans, with few or no side effects.